Stx12 and CCDC22 bind to the same region of VPS33B, which is essential for α-granule biogenesis. (A) Model of the human VPS33B 3-dimensional structure based on the human VPS33A crystal structure (PDB code: 4BX9).⁵⁰ Deletions in the helical hairpin region of VPS33B are indicated in pink (Δ314-336) and yellow (Δ308-345). (B) Gel filtration analysis of purified WT or mutants Twin Strep-VPS16B/His-VPS33B complexes. (C) VV beads containing WT or the different VPS33B deletions were tested for their ability to pulldown GST-Stx12 SNARE or His-CCDC22 CC. Bound proteins were analyzed by IB using antibodies against GST and CCDC22 as well as by SDS-PAGE (Coomassie). (D) IB analysis of total cell extracts of VPS33B KO cells expressing GFP, GFP-VPS33B WT, or GFP-VPS33B Δ314-336. (E) Quantification of the vWF and VPS16B IB band intensities from the experiment shown in panel D. The ratio of the band intensity of either VPS33B Δ314-336 or VPS33B WT samples over the VPS33B WT sample for both vWF and VPS16B proteins were compared, and statistical significance was determined via unpaired, 2-tailed Student t test (n = 2). ns, not significantly different from WT.